INTRODUCTION• Chromo = color , + soma = body.

• First described by Strausberger (1875).

• Term ‘Chromosome’ coined by Waldeyer (1888).

• Appear rod-shape during mitotic metaphase.

• Morphology changes with the stage of the cell division.

CHROMOSOME SIZE• Chromosome size changes drastically with the stage of

cell division.

• Measured during mitotic metaphase.

• Range varies from o.5 micron to 32 micron.

• Chromosome are longer in plants than in animals.

• Chromosomes are longer in monocot plants than in dicot plants.

CHEMICAL COMPOSITION

DNA = (30-40 % ) .

Protein = (50- 65 % ).

RNA = mostly rRNA .

FunctionContains genes, provides the genetic information.

Protect genetic material from chemical and mechanical damages.

Regulation of gene function.

CHROMOSOME STRUCTURE

• Components of chromosomes are Chromatid. Centromere. Telomere. Secondary constriction and Chromomere.

CHROMOSOME STRUCTURE:

(1) Chromatid – one of the two identical parts of the chromosome after S phase. (2) Centromere – the point where the two chromatids touch, and where the microtubules attach. (3) Short arm. (4) Long foot

HISTORY

• Chromosome walking was developed by Welcome Bender, Pierre Spierer, and David S. Hogness in the Early 1980's.

• The paper in which they first describe the technique is: Bender W, Spierer P, and Hogness DS. (1983)

• Chromosomal Walking and Jumping to Isolate DNA from the ACE and rosy Loci and the Bitho; Mol Biol. 168: 17-33, rax Complex in Drosophila melanogaster J

• In 1981, C. Weldon Jones and Fortis C. Kafatos describe the use of chromosomal walks to map Chorion genes in Silkmoths

Chromosome Walking and Chromosome Walking and Characterization of Chromosome Characterization of Chromosome

SegmentsSegments

• When a probe is used for the identification of a gene sequence in a genomic library, the probe may hybridize with a number of clones, each carrying a part of a large gene fragmented during preparation of genomic library

• we obtain partial digests from the genome, different genomes (from large number of cells) may give fragments which have overlapping sequences, because sites cleaved in different genomes of the same organism, will differ being random.

• Since none of these fragments may have its entire sequence represented in the probe, overlapping sequences may be used to construct the original genomic sequence.

• Identification of fragments with an overlapping sequence may be a key to the reconstruction or characterization of large chromosome regions. This is achieved by the technique popularly called chromosome walking.

• In short-- technique for identification and isolation of contiguous sequences of genomic DNA

Technique of chromosome walking..

(i) from the genomic library select a clone of interest (identified by a probe) and subclone a small fragment from one end of the clone (there is a technique available to subclone a fragment from the end);

Technique of walking through successive hybridization between chromosome overlapping genomic clones.

(ii) the subcloned fragment of the selected clone may be hybridized with other clones in the library and a second clone hybridizing with the subclone of the first clone is identified due to presence of overlapping region;

Technique of walking through successive hybridization between chromosome overlapping genomic clones.

(iii) End of the second clone is then subcloned and used for hybridization with other clones to identify a third clone having overlapping region with the subcloned end of the second clone;

Technique of walking through successive hybridization between chromosome overlapping genomic clones.

(iv) third clone identified as above is also subcloned and hybridized with clones in the same manner and the procedure may be continued

Technique of walking through successive hybridization between chromosome overlapping genomic clones

(v) Restriction map of each selected clone may be prepared and compared to know the regions of overlapping so that identification of new overlapping restriction sites will amount to walking along the chromosome or along a long chromosome segment.

Technique of walking through successive hybridization between chromosome overlapping genomic clones

Technique of walking through successive hybridization between chromosome overlapping genomic clones.

Use of Chromosome walking Use of Chromosome walking

• chromosome walking was used to identify the surrounding sequence of a particular "unknown" gene which was known to lie between two markers.

Use of chromosome walking in discovery of single-nucleotide polymorphism in noncoding regions of a candidate actin gene in Pinus radiata.

• This technique can be used to analysis diseases, such as cystic fibrosis, to look for mutations.

Chromosomal Walking to Clone the Cystic Fibrosis Gene

• The first thing you need to do is create two genomic libraries of the same DNA but each library used a different restriction enzyme, such as EcoR I and Sal I.

One genome is used to prepare two genomic libraries with two different restriction enzymes.

• Next, must have a probe that is linked to your favorite gene (YFG). In this example, we want to clone the cystic fibrosis gene (CF). You would screen a genomic library with this probe and isolate a piece of DNA that binds to your probe.

If you cloned an EcoR I restriction fragment from the genomic library (let's say it is fragment #3 of many possible fragments) that binds to a particular probe (called MET) that is linked to CF.

• You want to slide down the chromosme from MET towards CF so you can clone and sequence CF. D7S8 is another RFLP marker located on the other side of CF so CF is located between MET and D7S8

One EcoR I fragment that binds to the probe MET. Located at the other end is a second RFLP marker called D7S8 and CF is located somewhere between

these two markers.

• To clone CF, you will employ chromosomal walking to take baby steps towards CF, starting with the EcoR I restriction fragment #3 you just cloned. Now, you need to generate a restriction map of EcoR I fragment #3. You must digest the Eco RI restriction fragment with multiple restriction enzymes and analyse the results on an agarose gel as shown here:

agarose gel that contains the restricted DNA

If you then performed a Southern blot with this gel and used the original MET probe that allowed you to isolate the EcoR I restricion fragment, you might see the following resutls on an X-ray film:

X-ray film obtained when the Southern blot is probed with MET.

Construct the following restriction map that also indicates where the probe binds. NNote: two slightly different restriction maps could be generated from these data but this one is fine for our purposes..

One possible restriction map, given the data from figures 1 and 2 above.

The next task at hand is to isolate the 2.5 kb Sal I - EcoR I fragment and use it as your second probe on the Sal I genomic library because this 2.5 kb piece is the DNA fragment furthest from the MET marker and therefore must be closer to CF.

Used screen the Sal I genomic library that used identical DNA but was digested with the restriction enzyme Sal I instead of EcoR I. Because probe #2 is flanked on the left by a Sal I site, new fragment that has Sal sites on both ends and binds to the second probe will extend towards the right (in the direction of CF) as shown:

How use the first genomic DNA clone to generate a second probe that takes one step in the direction of CF.

When have cloned a Sal I fragment that binds to probe #2, need to figure out its restriction map the same way for the EcoRI fragment #3 above. This process continues until you reach D7S8.

The final product pf a chromosomal walk is a series of overlapping restriction maps starting at your original probe (MET) and extending to D7S8. The final combined restriction map, and the overlapping fragments, might look like this

How a series of overlapping pieces of genomic DNA has been isolated from alternate genomic libraries.

Involving only 5 steps for the walk.

Now a restriction map spanning the area of interest, use a number of different methods to determine which fragment contains the CF gene. IIt is very simple to sequence the CF gene and continue your analysis.

CF, chromosomal walking can be used to move from any location on a chromosome towards another gene you want to clone but know very little about, including its sequence.

ReferencesReferences

Singh B. D. 2007. Biotechnology Expanding Horiozone. Kalyani publishers.

E J Gardner, M J Simmons, D P Snustad, 2005. Principles of genetics(8 editn) John Wiley & Sons (Asia) Pte. Ltd. Singapore.

B D Hames, & N M Hooper, Instant Note Biochemistry 2000. (2nd editn) BIOS Scientific Published Limited.

Thank you…Thank you…